Conjugating biologically active proteins to polymers has been shown to improve the circulating life of the administered protein and to reduce its antigenicity and immunogenicity. For example, U.S. Pat. No. 4,179,337 discloses the use of PEG or polypropylene glycol coupled to proteins to provide a physiologically active non-immunogenic water soluble polypeptide composition. Conjugates are formed by reacting a biologically active material with a several fold molar excess of a polymer which has been modified to contain a terminal linking group.
A variety of means have been used to attach polyethylene glycol molecules to the protein. For example, U.S. Pat. No. 5,932,464 and U.S. Pat. No. 5,990,237 disclose methods for coupling polyethylene glycol to a biomaterial. Generally, polyethylene glycol molecules are connected to a protein via a reactive group found thereon. Amino groups, such as those on lysine residues or at the N-terminus, as well as thiol groups on cysteine, or other reactive groups on protein surface, are convenient for such attachment. For many biologically active materials, however, the conjugation process is accompanied by several complications. Firstly, it is not always specific with regard to attachment sites. Secondly, loss of biological activity is often caused by the conjugation reaction. For example, if too much of the activated polymer is attached to the target protein or polypeptide, biological activity can be severely reduced or lost. Furthermore, if the wrong linker joining the polymer to the protein is used, or if an insufficient amount of polymer is attached to the target, the therapeutic value of the resultant conjugate is limited. Often, such conjugates do not demonstrate enough of an increase in the circulating life to compensate for the loss in bioactivity. Problems can also result when a therapeutic moiety's active site (i.e. where groups associated with bioactivity are located) becomes sterically blocked as a result of the polymer attachment. Accordingly, the outcome of a protein conjugation process is unpredictable in nature.
Cholinesterases are important proteins. Acetylcholinesterase (AChE, EC 3.1.1.7) plays a pivotal role in the cholinergic system where it functions in the rapid termination of nerve impulse transmission. The function of the related enzyme butyrylcholinesterase (BChE, EC 3.1.1.8) is yet unknown, nor is its specific natural substrate known, but it is capable of hydrolysing acetylcholine. It has been suggested that BChE acts as an endogenous scavenging enzyme important for the detoxification of natural poisons [Massoulie, J., et al., (1993) Prog. Brain Res. 98, 139-146]. The high reactivity of these enzymes toward organophosphorus (OP) compounds, makes exogenous cholinesterase an effective therapeutic agent in the prophylaxis and treatment of OP-poisoning. Indeed the successful exploitation of the scavenging potential of various forms of cholinesterases which include fetal bovine AChE [Maxwell, D. M., et al., (1992) Toxicol. Appl. Pharmacol. 115, 44-49], human BChE [Raveh, L., et al., (1993) Biochem. Pharmacol. 45, 37-41], equine BChE [Broomfield, C. A., et al., (1991) J. Pharmacol. Exp. Ther. 259, 683-698] has been demonstrated in rodents [Raveh, L., et al., ibid] and in non-human primates [Broomfield, C A, et al., ibid; Maxwell D M et al., ibid] and even for treatment of humans exposed to organophosphate pesticides [Cascio, C., et al., (1988) Minerva Anestesiol. 54, 337-338].
The use of ChE as a biological scavenger requires sources for large quantities of purified enzyme and depends on the retention of the enzyme in the circulation for sufficiently long periods of time. Production of AChE in various expression systems is known. However, the successful application of recombinant ChE as a bioscavenging agent of therapeutic value requires its retention within the circulation for appreciable periods of time. Examination of the pharmacokinetic profile of various recombinant AChEs demonstrates that these are eliminated from the circulation rapidly, displaying mean residence time values (MRT) of 5-100 minutes [Kronman, C., et al., (1992) Gene 121, 295-304; Chitlaru, T., et al., (1998) Biochem. J. 336, 647-658], and therefore do not meet the requirements for OP bioscavenging in their non-modified state.
It would therefore be desirable to be able to provide ChEs that exhibit improved retention within the circulation, which could be used as therapeutic bioscavenging agents. It has now been surprisingly found, and this is an object of the present invention, that it is possible to provide such improved modified ChEs, by a modification made using polyethylene glycol groups attached to the lysine moieties located on the ChEs.
It is thus an object of the invention to provide such modified ChEs which exhibit excellent and unprecedented circulatory longevity.
It is another object of the invention to provide a beneficial extension of circulatory residence by polyethylene glycol appendage, which overrides the various deleterious factors which contribute to the rapid clearance of recombinant AChEs, allowing long-term circulatory residence of recombinant AChE molecules which are devoid of glycans, or contain glycans devoid of sialic acid capping, or do not assemble into multimeric forms.
It is a further object of the invention to provide pharmaceutically-compatible AChEs from a wide variety of sources, including those which display suboptimal post-translation processing.
It is a further object of the invention to provide a novel use of the modified AChEs of the invention in the prophylactic or acute treatment of OP poisoning.
Other objects and advantages of the invention will become apparent as the description proceeds.